Our studies on microRNAs in the cornea have made excellent progress in the past year. miRNAs are a class of small endogenous noncoding RNAs that posttranscriptionally down regulate gene expression and play important roles in embryonic development, differentiation, and cancer. To evaluate the roles of miRNAs on corneal and lens development, we generated transgenic mice whose developing lens and corneal epithelium were conditionally targeted for Dicer gene, an essential enzyme for miRNA biogenesis. The resultant Dicer conditional null mice (Le-Cre/fDicer) had severe microphthalmia with no discernible lens. Removal of miRNAs induced cell death in the developing lens with severe lens dystrophy by Embryonic Day 18.5 (E18.5). In contrast, lack of Dicer gene did not induce cell death in the developing corneal epithelium. The morphology of the developing corneal epithelium in the conditional null mice showed no abnormality at birth, although expression of Keratin 12 (Krt12), one of the corneal epithelial differentiation markers, was greatly reduced. The mature corneal epithelium of the 6-week-old conditional null mice was less stratified as compared to the controls and there was no identifiable limbal region. Our observations suggest that miRNAs play a different role in corneal and lens development: miRNAs may have major contributions to maturation and specification of corneal epithelium, while playing essential roles in cellular proliferation and survival during lens morphogenesis. An additional goal of this study is to identify specific miRNAs that are critical for corneal and lens development. We have previously identified miR-184 as one of the most abundant miRNAs in the cornea, and have shown its expression is highly cornea/lens-specific. To investigate the regulation and function of this miRNA, we performed 5- and 3- RACE analyses, identified the transcriptional start site and the polyadenylation site, and demonstrated that the primary transcript of miR-184 is about 1Kb in length. We found that this 1-kb primary transcript could be transcribed and processed to form mature miR-184 in cultured cells when cloned into an expression vector. In addition, the 2kb upstream sequence of the identified transcript displayed promoter activity in cell culture. Algorithmic prediction has identified in the 2kb upstream sequence the potential binding sites for corneal and lens transcription factors, including Pax6, Oct1, and Klf. This study represents the first identification and characterization of a mouse miRNA promoter in cornea and lens. We have furthered our studies of the cornea by investigating the corneal phenotype of mice with targeted deletion of the PdLim2 gene. Pdlim2 is an E3 ubiquitin ligase, expressed at high levels in corneal epithelium, which contains an N-terminal PDZ motif and a C-terminal LIM domain. Previous tudies have shown that Pdlim2 interacts with cytoskeletal proteins, co-localizes with the actin cytoskeleton, and is required for epithelial migration. Analysis of Pdlim2 knockout (KO) mice showed that about 70% have corneal abnormalities, including loss or disruption of the basement membrane and neovascularization at the stromal/epithelial interface. Corneal erosions were also frequently seen. Comparing the corneal protein expression profiles of wild type and KO mice by 2D differential fluorescence gel electrophoresis identified 39 spots with more than 2 fold differences. Sequencing and functional annotation of 13 spots revealed significant clustering in the categories of cytoskeletal proteins and chaperones. Immunoblotting confirmed a 3 to 5 fold increase of certain proteins from both categories in KO mice, including -actin, destrin, -tubulin, stathmin 1, tubulin specific chaperone BznA-crystallin, and -crystallin. Analysis of corresponding mRNAs suggests that protein levels are post-transcriptionally controlled, possibly by ubiquin-mediated proteolysis. Although PdLim2 is localized in the nucleus in most cells, in the corneal epithelium it is entirely localized in the cytoplasm, associated with the actin cytoskeleton. TIRF (total internal reflectance fluorescence) microscopy of transfected cells revealed that GFP-Pdlim2 localized to sites of cell-matrix adhesion and actin attachment. Moreover, a GFP-Pdlim2 fusion protein co-immunoprecipitated with -actinin and actin in transfected corneal epithelial cells. GFP fusions with isolated PDZ-, Middle-, and LIM- domains of Pdlim2 showed that each of these regions bound to -actinin and actin, but much less strongly than the full length protein. Transfection of the isolated domains of Pdlim2 identified an important role for the middle domain in the cytoplasmic localization of Pdlim2: constructs lacking this region were found primarily in the nucleus. Since the middle domain also contains numerous potential phosphorylation sites, we are exploring the possibility that phosphorylation may regulate the nuclear-cytoplasmic distribution of PdLim2. Together these findings suggest a role for Pdlim2 as cytoskeletal associated adaptor protein involved in maintaining the normal structure, morphology, and integrity of the corneal epithelium. (Studies of PdLim2 in the cornea were previously reported as a part of the project EY-000238, Signal Transduction in Epithelial Cells of the Lens and Cornea.) The corneal-enriched expression of aldehyde dehydrogenase 3a1 (ALDH3a1) in the mouse has been explored by assessing the promoter activity of ALDH3a1 promoter constructs in tissue culture and transgenic mice. Genomic sequences residing 8kb upstream of the ALDH3a1 transcriptional initiation site, all 10 introns, and the 3untranslated region of the ALDH3a1 gene have been examined. Sequences that differentially activate and repress ALDH3a1 promoter activity in cornea, skin, lens, liver, stomach, lung, and brain have been identified. Of note, the first intron can enhance promoter activity in cornea, skin, stomach and lens, but also contains a region that is able to abolish promoter activity under certain conditions. Previously, we showed that several transcription factors including PAX6, OCT1, and KLF4 regulate the activity of the ALDH3a1 promoter. To explore the role of Pax6 in the cornea, an examination of the ocular phenotype of transgenic mice overexpressing Pax6 using the ALDH3a1 promoter has been completed. Corneal abnormalities include an immune infiltrate, neovascularization, and changes in the number of corneal epithelial cell layers, cell shape, and general morphology of the cornea epithelium. Microarrays identified differentiall expression of genes involved in the immune system, vascularization, and epithelial differentiation in the Pax6 transgenic corneas. Altered expression of chitinase-like proteins, toll-like receptors, and many chemokine ligand and receptors suggested activation of an immune response. Genes involved in blood vessel formation, including angiopoietin-like 2, angiomotin, and VEGF-C, were also differentially expressed;however, expression of Flt1, the soluble VEGF receptor shown to play a major role in corneal vascularization remained unchanged. Finally, many differentiation markers, a family of small proline-rich proteins, and several components of the Wnt pathway showed changes in gene expression. To determine whether these genes are targets of Pax6, a promoter region from a gene representing each of these functional categories was fused to a reporter gene and analyzed by transfection. Cotransfection of Pax6 activated the promoters of Chi3l4 and WIF1 in COS-7 cells, but had no effect on Flt1, corroborating results observed by microarray. Finally, the loss of corneal epithelial markers such as keratin12 is accompanied by a reduction in the amount of Pax6 protein and a loss of nuclear Pax6 supporting a role for Pax6 in corneal differentiation.